Process for the production of isoxazolyl penicillins

ABSTRACT

WHEREIN R1, R2 and R&#39;&#39; are as defined above; then subjecting the compound of Formula V to dephenylacetylation; and eliminating R&#39;&#39; when R&#39;&#39; is a said easily eliminated group.   A chemical process for the production of isoxazolyl penicillins and nontoxic salts and lower alkyl esters thereof having the formula REQUIRES NO MICROBIOLOGICAL DEACYLATION OR FERMENTATION TO PRODUCE 6-AMINOPENICILLANIC ACID, BUT INSTEAD COMPRISES REACTING A PENICILLIN G ester having the formula WHEREIN R&#39;&#39; is a protective group for carboxyl group and is selected from the group consisting of lower alkyl and a group which, when subjected to an elimination reaction, can be easily eliminated without having any detrimental effect on the structure of penicillin, which a chlorinating agent in the presence of a tertiary organic base to obtain an imide chloride groupincorporated compound having the formula WHEREIN R&#39;&#39; is as defined above; reacting the compound of Formula III with an isoxazol carboxylate having the formula WHEREIN M is a metal atom, and R1 and R2 are as defined above, to obtain a diacyl penicillin ester having the formula

United States Patent A chemical process for the production of isoxazolylpenicillins Abetal,

1541: PROCESS FOR" THE PRODUCTION OF ISOXAZ OLYL PENICILLYINS Abe,Yoshida;

[721- Inventors: Jinnosuke' Tetsuo Watanabe, Yokohama-shi; Teruo Take,v

' Tagata-gun; Kentaro Ftdimoto, Tagatagun; Tadashiro Fujii, Tagata-gun;Kazunari Takemura, Tagata-gun; Kazuyoshl Nlshiie, Numazu-shi; SakneSatoh, Sakaishi; Tatlashl Koide; .Yasue Hotta, both of lagata-gun, allof Japan 117111 Aimignec:

I I I Japan 7 I 221 Filed: Sept. 24, 1969 21 1" Appl. No.: 860,812

s21 'u.s. c|. ..260/239.1,424/27 1" Primary Examiner-Nicholas S. RizzoAttorney-Youngand Thompson ABSTRACT and nontoxic salts and lower alkylesters thereof having the formula requires no microbiologicaldeacylation or fermentation to produce 6-aminopenicillanicacid, butinstead comprises 4 1 June 6, 1972 reacting a penicillin G ester havingthe formula wherein R. is aprotective group for carboxyl group and is aselected from the group consisting of lower alkyl and a group which,when subjected to an elimination reaction, can be easily eliminatedwithout having any detrimental effect on the structure of penicillin,which a chlorinating agent in the presence of a tertiary organic base toobtain an imide chloride group-incorporated compound having the formulaS CH; CH:C=NCHC{I .\C/

61 l y 1 pm,

' o= -N,-- H-COOR' 111 wherein R" is as defined above; reacting thecompound ofl-ormula ill with an isoxazol carboxylate having the formulawherein M is a metal atom,. and R and R are as defined above, to obtaina diacyl penicillin ester having the formula wherein R,, R and R are asdefined. above; then subjecting the compound of Formula V todephenylacetylation; and eliminating R when R is a said easilyeliminated group.

6 Claims, No Drawings PROCESS FOR THE PRODUCTION OF ISOXAZOLYLPENICILLINS This invention relates to a process for production ofisoxazolyl penicillins having the formula wherein R represents hydrogenor a lower alkyl group, and R and R are the same or different and eachrepresents hydrogen 15 or a halogen atom.

More particularly, the invention pertains to a process for preparingisoxazolyl penicillins represented by said Formula I, or nontoxic saltsor loweralkyl esters thereof, characterized in that a penicillin G esterhaving the formula wherein R is a protective group for carboxyl groupand represents a lower alkyl group or a group which can be easilyeliminated without having any detrimental effect on the structure ofpenicillin, is reacted with a chlorinating agent in the presence of atertiary organic base to obtain an imide chloride group-incorporatedcompound having the formula 0: H-COOR' (III) whiirhi i lilirlii'hlbbvethe compound ti fibrmrlilifi is reacted with an isoxazol carboxylaterepresented by the formula defined previously, to obtain a diacylpenicillin ester represented by the formula wherein R R and R areasdefined previously; and then the 65 compound of Formula V is subjectedto dephenylacetylation, and in case R in Formula V is a group capable ofbeing easily eliminated without having any detrimental effect on thestructure of penicillin, the resulting product is so treated as toeliminate the protective group for carboxyl group.

Heretofore, isoxazolyl penicillins have been prepared in such a mannerthat 6-aminopenicillanic acid prepared by microbiological deacylation ofpenicillin G or V obtained by fermentation, or 6-aminopenicillanic acidobtained by direct fermentation, is acylated according to such anacylation 75 process as the acid halide process, acid anhydride process,mixed acid anhydride process, active ester process or process using acondensing agent (dicyclohexyl carbodiimide or carbonyl imidazole) asfor example in U.S. Pat. No. 2,996,501. According to these processes,however, the amphoteric compound 6-aminopenicillanic acid should beisolated from the fermentation liquor and, due to the troublesomeness ofthe isolation step, the lowering of yield has been unavoidable.

The present invention is characterized by the fact that isoxazolylpenicillins are prepared chemically without using 6- aminopenicillanicacid but using as a starting material inexpensive penicillin Gobtainable by fermentation.

An object of the present invention is to provide a novel process forpreparing isoxazolyl penicillins which is entirely different from theconventional processes for preparing semisynthetic penicillins byacylating -aminopenicillanic acid.

In the present invention, penicillin G obtained by fermentation is usedas a starting material. However, the penicillin G is 0 not used as it isbut is used in the form of an ester such as penicillin G esterrepresented by Formula II (hereinafter said ester will be referred to asthe penicillin G ester II") which is formed by introducing a protectivegroup into the carboxyl group of penicillin G.

The carboxyl group of penicillin G is protected so that no side reactionwill be brought about in the subsequent reaction, i.e. the reaction witha chlorinating agent.

The above-mentioned introduction of a protective group is carried out byintroducing into a potassium or sodium salt of penicillin G obtained byfermentation such a protective group as is used ordinarily in thesynthesis of peptides. Examples of protective groups which may beintroduced are methyl, ethyl, t-butyl, p-methoxybenzyl, benzyl,p-nitrobenzyl, benzhydril, phenacyl, p-bromophenacyl and trimethylsilylgroups. Generally, however, the protective group introduced should beeliminated in the final step and therefore it is desirable to select agroup capable of being easily eliminated without having any detrimentaleffect on the structure of penicillin. Favorable results are obtained bythe use of such protective groups as, for example, benzyl, p-nitrobenzyland benzhydril groups which can be easily eliminated by catalyticreduction, or phenacyl and p-bromophenacyl groups which can beeliminated with sodium thiophenoxide or thiophenolate.

Generally, penicillins are unstable to acids and alkalis. Accordingly,when such a protective group which is eliminated by acid or alkalihydrolysis, e.g. a methyl, ethyl, tbutyl or pmethoxybenzyl group, hasbeen introduced, the elimination thereof is difficult because thedestruction of the structure of the penicillin is brought about at thetime of the elimination reaction. in the present invention, however,even when the final products, i.e. isoxazolyl penicillins, are in theform of esters having protective groups, the object of the invention canbe accomplished without eliminating the protective groups insofar as theprotective groups are those of the kind which do not havepharmacologically detrimental efiects. Examples of such protectivegroups are lower alkly groups such as methyl, ethyl and the like groups.

In accordance with the present invention, the penicillin G ester II isreacted with a chlorinating agent to obtain an imide chloridegroup-incorporated compound represented by Formula III (hereinafter saidcompound will be referred to as the imide chloride III"). The abovereaction is an application of a process in which an N-mono-substitutedcarboxylic acid amide is treated with a chlorinating agent to produce acorresponding imide chloride group-incorporated compound, and it is mostpreferable to adopt a process in which the reaction is effected, in the.presence of a tertiary organic base, using such chlorinating agents asPCl PCl POCI COCI SOCl etc., PC], being preferred. See South AfricanPat. No. 67/2927.

In the above-mentioned reaction, it is preferable to use a suitable dryorganic solvent. This solvent is preferably selected with regard to thefact that it should not substantially detrimentally affect the reaction;that it should be able to solubilize the penicillin G ester II; and thatit should be an organic solvent immiscible with water because, after thereaction, byproducts formed are removed by washing. For example,benzene, toluene, chloroform, dichloromethane, dichloroethane, ethylether or isopropyl ether are advantageously used.

If PCl for example, is used in the above reaction as the chlorinatingagent, POCl and HCl are necessarily formed. In case a hydrochloride ofthe tertiary organic base has precipitated in the reaction liquid, it isfirst removed by filtration and, since the imide chloride Ill obtainedis relatively stable to water, POCl can be removed from the reactionliquid by washing with an aqueous weakly alkali solution, e.g. anaqueous sodium or potassium bicarbonate solution. It is desirable thatthe above washing operation be carried out as quickly as possible and,after the washing, that the reaction mother liquor be immediately driedby addition of anhydrous sodium sulfate or magnesium sulfate.

In view of the fact that compounds having imide chloride groups areunstable in water, in general, it is very surprising that the imidechloride III obtained by the aforesaid reaction is relatively stable inwater. Further, it is extremely favorable that, by the above-mentionedoperation, a reaction mother liquor containing the imide chloride IIIcan be obtained in a stable state.

In the above-mentioned reaction mother liquor, unreacted tertiaryorganic base remains as it is. This base can be easily removed from thereaction mother liquor as a water-soluble acid addition salt by washingthe liquor with a dilute acid. However, the imide chloride III isunstable in acids and therefore it is desirable that said base not beremoved. As the tertiary organic base employed in the present invention,therefore, the use of such a weak base as pyridine, for example, givesfavorable results. This is because in the subsequent reaction, i.e. inthe step where the imide chloride III is reacted with an isoxazolcarboxylate represented by Formula IV (hereinafter said carboxylate willbe referred to as the carboxylate IV) to form a diacyl penicillin esterrepresented by Formula V (hereinafter said ester will be referred to asthe diacyl penicillin ester V), if a strongly basic tertiary organicamine, e.g. triethylamine or the like, is present, there is danger thata stereochemical rearrangement (epimerization) may take place in thehydrogen atom at the 6-position of the penicillin nucleus; but in thepresence of such a weak base as pyridine, there is little danger ofepimerization.

The imide chloride III contained in the above-mentioned reaction motherliquor may be reacted as is, without any further purification, with thecarboxylate IV. Alternatively, it may be reacted with the carboxylate IVafter concentrating the reaction mother liquor and dispersing theconcentrate in another suitable solvent, e.g. benzene, toluene,chloroform, dichloromethane, dichloroethane, ethyl ether, isopropylether, tetrahydrofuran or dioxane. However, an aqueous solutiontreatment is effected after the reaction and therefore it isadvantageous to use a water-immiscible solvent.

Examples of the carboxylate IV to be reacted with the imide chloride IIIare metal salts, such as potassium, sodium, lithium and silver salts, of3-phenyl-5-methyl-isoxazol-4-carboxylic acid, 3-( 2 '-chlorophenyl)--methyl-isoxazol-4-carboxylic acid,3-(2-bromophenyl)-5-methyl-isoxazol-4-carboxylic acid, 3-(2'-fluorophenyl )--flurophenyl)-5-methyl-isoxazol-4- carboxylic acid,3-(2',6-dichlorophenyl)-5-methyl-isoxazol-4 -carboxylic acid,3-(2-chloro-6'-fluorophenyl)-5-methylisoxazol-4-carboxylic acid and3-(2-bromo-6-chlorophenyl)- 5-methyl-isoxazol-4-carboxylic acid. Thecarboxylate IV may be added directly to the reaction mother liquorcontaining the imide chloride III or may be used in the form of asuspension in the same solvent as in the reaction mother liquor. In thiscase, if a strongly basic tertiary organic base such as triethylamine orthe like is used in place of the carboxylate IV, there is danger thatthe resulting diacyl penicillin ester V may cause epimerization of thehydrogen atom in the 6-position of the penicillin nucleus, as mentionedpreviously. However,

when a metal salt such as a potassium, sodium, lithium or silver salt isused, said epimerization can be prevented or the degree of epimerizationcan be greatly reduced.

Theoretically, the quantitative proportion of the carboxylate IV isequimolar to the imide chloride III. After the reaction, however, theremoval of unreacted carboxylate IV is easier than the removal ofunreacted imide chloride III. Ordinarily, therefore, the carboxylate IVis used somewhat in excess.

The above-mentioned reaction progresses readily at normal temperatureand therefore no particular heating is necessary. However the reactionmixture may be heated to about 40 to 50 C. if the reaction rate isundesirably slow; but overheating or rapid heating should be avoidedbecause of the decomposition of imide chloride III or diacyl penicillinester V.

By the above reaction, there is obtained the diacyl penicillin ester V.In separating this reaction product, the reaction liquid is washed withan aqueous dilute acid solution, an aqueous dilute alkali solution, andwater, in this order, to remove remaining tertiary organic base, e.g.pyridine or the like, and unreacted carboxylate IV, and then thereaction mother liquor is concentrated and is subjected to silicagel-column chromatography using a solvent of benzene-ethyl acetate orthe like system, whereby only the desired diacyl penicillin ester V isinitially eluted. When the eluted fraction is subjected to freeze-dryingor the like, the product can be easily isolated.

The thus-obtained diacyl penicillin ester V can be subjected, withoutany further purification, to the subsequent reaction, i.e. to thedephenylacetylation reaction.

In the present invention, the phenylacetyl group of the diacylpenicillin ester V is eliminated to obtain an isoxazolyl penicillinester represented by the formula wherein R R and R are as definedpreviously (hereinafter said ester will be referred to as the isoxazolylpenicillin ester VI). The elimination of said group may be effectedaccording to any of such processes as aminolysis, solvolysis,hydrolysis, splitting by thiophenolate and the like. Of these, theprocesses using an aminolyzing agent and splitting by the use ofthiophenolate are the most preferable dephenylacetylation processes.

The above reaction is such that, of the two acyl groups, i.e.phenylacetyl and isoxazolyl groups, which have been bonded to thenitrogen atom in the 6-position of penicillanic acid of the diacylpenicillin ester V, only the former is entirely selectively cleaved.

In the most preferable reaction mentioned above, a primary organicamine, a secondary organic amine, ammonia, primary aliphatic amine orthe like is used as the aminolyzing agent. In this reaction, only thebond on the side of phenylacetyl group is cleaved, because the bond onthe side of isoxazolyl group is stable, and an N-substitutedphenylacetamide is formed as byproduct. This reaction sometimes bringsabout, depending on the kind of aminolyzing agent, the epimerization ofthe hydrogen atom in the 6-position of the penicillin nucleus, or thecleavage of ,B-lactam bond of the penicillin nucleus, simultaneouslywith the aminolysis. However, preferable aminolysis conditions can beensured by the selection of suitable aminolyzing agent, reaction solventand the like.

In the above reaction, the solvent is desirably so selected that it willnot have any detrimental effect, such as epimerization or decompositionof the B-lactam ring, on the reaction; that it will be able tosolubilize the isoxazolyl penicillin ester VI; and that it will be anorganic solvent immiscible with water because, after the reaction,unreacted aminolyzing agent, e.g. a primary organic amine, is removed bywashing. For example,

benzene, toluene, chloroform, dichloromethane, dichloroethane, ethylacetate, butyl acetate, ethyl ether or isopropyl ether may beadvantageously used.

As the aminolyzing agent, it is advantageous to use a primary organicamine, e.g. a cylcoalkylamine such as cyclohexylamine, benzylamine, aphenylaklyamine such as B-phenylethylamine, or a primary aliphatic aminesuch as ethylamine, n-butylamine, n-propylamine, n-pentylamine,n-hexylamine, n-heptylamine, n-octylamine, caprylamine, laurylamine,myristylamine, palmitylamine, stearylamine or the like. In the lastcase, the greater the number of carbon atoms, the less the danger ofepimerization and the less the decomposition of isoxazolyl penicillinester VI. Accordingly, an amine having four to 20 carbon atoms ispreferably used and a straight chain aliphatic amine is advantageouslyused. In the case of branched chain aliphatic amines, it is better notto have a side chain at the a-position of the carbon chain. Among theseamines, straight chain higher aliphatic amine having more than 12 carbonatoms such as laurylamine, myristylamine, palmitylamine, stearylamine orthe like are the most advantageously used. Theoretically, thequantitative proportion of the aminolyzing agent may be equimolar to thediacyl penicillin ester V. However, the aminolyzing agent is more easilyremoved than unreacted diacyl penicillin ester V and hence is ordinarilyused in an amount of one to two times the moles of the diacyl penicillinester V.

The above-mentioned reaction progresses even at low temperatures andtherefore no heating is necessary unless the reaction of the diacylpenicillin ester V with a primary organic amine is undesirably slow. Incase a secondary organic amine is used, however, the reaction is slowand therefore it is sometimes necessary to heat the reaction mixture.

The dephenylacetylation reaction by solvolysis may be effected by use ofa lower aliphatic alcohol, e.g. methanol or ethanol, or a hydrated loweraliphatic alcohol, e.g. hydrated methanol or hydrated ethanol.

The dephenylacetylation reaction by hydrolysis may be carried out bytreatment with an acid or an alkali in a hydrophilic organic solventcapable of dissolving the diacyl'penicillin ester V such as, forexample, dimethylformamide, acetone, dioxane or tetrahydrofuran.

According to the above-mentioned reaction, the isoxazolyl penicillinester VI is obtained. In separating this reaction product, the reactionliquid is washed with a dilute acid and water, in that order, wherebyunreacted aminolyzing agent, e.g. a primary organic amine, can beremoved from the reaction liquid as a water-soluble acid addition saltto isolate the product.

When a primary aliphatic amine is used as an aminolyzing agent, therewill be present isoxazolyl penicillin ester VI, byproduct aliphaticgroup-substituted phenylacetamide, and unreacted primary aliphaticamine. When eliminating isoxazolyl penicillin ester VI therefrom, ifprimary aliphatic amine is formed as a water soluble acid addition saltsuch as lower aliphatic amine, the reaction mixture should be washedwith dilute acid and water respectively to remove primary aliphaticamine which forms a water soluble acid addition salt.

The said reaction mixture is charged to a column of silica gel oralumina and then eluted with a suitable solvent such asbenzene-chloroform or benzene-ethylacetate to separate isoxazolylpenicillin ester VI readily therefrom.

The isoxazolyl penicillin ester VI may be isolated, if necessary;however, it is not necessary to separate it from the reaction mixture,and the condensate or lyophilizate thereof may be used in a subsequentreaction, e.g. removal of the protective group of isoxazolyl penicillinester VI.

In'the next step, the protective group for the isoxazolyl penicillinester VI is eliminated to obtain an isoxazolyl penicillin represented byFormula I (hereinafter said penicillin will be referred to as theisoxazolyl penicillin I). In the present invention, however, when R inFormula V is a lower alkyl group, the aforesaid elimination reactionresults in the destruction of the structure of the penicillin, andtherefore the isoxazolyl penicillin ester VI may be regarded as thefinal product without eliminating the protective group. When 'R inFormula V is a group which can be easily eliminated without having anydetrimental effect on the structure of penicillin, said group can beeliminated by a known process. That is, a benzyl, p-nitrobenzyl orbenzhydril group can be eliminated by catalytic reduction, and aphenacyl or p-bromophenacyl group can be eliminated by using sodiumthiophenoxide, or thiophenolate. In this elimination reaction, ifthiophenolate is used in more than two-molar excess with diacylpenicillin V, dephenylacetylation and deesterification occursimultaneously to produce the isoxazolyl penicillin free acid of FormulaI.

The catalytic reduction is effected by use of hydrogen gas in an inertorganic solvent, e.g. ethyl acetate, butyl acetate, chloroform,dichloromethane, benzene, toluene or ethyl ether, in the presence of apalladium-active carbon catalyst or the like. In this case, nosuperatmospheric pressure is required. The reaction progresses morequickly in the case of the p-nitrobenzyl group than in the case of thebenzyl group, and the former group is easily eliminated.

The isoxazolyl penicillin I obtained according to the abovementionedreduction reaction exists, in the reaction liquid, in the form of a freeacid. This isoxazolyl penicillin I can be isolated from the reactionliquid as a pharmacologically nontoxic salt according to known processesemployed in the case of penicillin G or V. For example, the reactionliquid may be neutralized with an n-butanol or butyl acetate solution ofpotassium or sodium 2-ethylhexanoate to convert the penicillin to asolvent-insoluble salt, followed by recovery. Alternatively, thereaction liquid is washed with an acid and water, in this order, isneutrally extracted with an aqueous potassium or sodium hydroxidesolution and is subjected to freeze-drying, and the penicillin isdirectly recovered in the form of a potassium or sodium salt or isprecipitated as a water-insoluble salt with an amine such asN,N-dibenzylethylenediamine and is then recovered.

In the next step, the elimination of the phenacyl or pbromophenacylgroup by means of sodium thiophenoxide is carried out according to theprocess of Sheehan et al., J. Org. Chem., 29 20068 (1964). When treatedwith sodium thiophenoxide in a suitable inert organic solvent such as,for example, dimethylformamide, tetrahydrofuran or dioxane, said groupis easily eliminated and, moreover, the isoxazolyl penicillin I isobtained as a sodium salt. Accordingly, when a solvent such as acetoneis added to the reaction liquid after completion of the reaction, thesodium salt of isoxazolyl penicillin I can be precipitated and theisoxazolyl penicillin I can be isolated with extreme ease. An isoxazolylpenicillin of Formula I in which R is a lower alkyl group, i.e. anisoxazolyl penicillin ester of Formula VI in which R is a lower alkylgroup, is in the form of a lower alkyl ester and hence does not form anynontoxic salt.

In the present invention, in accordance with the reaction betweenthiophenolate and diacyl penicillin V, of the two acyl groups, i.e.phenylacetyl and isoxazolyl groups, which have been bonded to thenitrogen atom in the 6-position of penicillanic acid, only the former isentirely selectively cleaved to produce isoxazolyl penicillin ester VI.Further, in case of the reaction with more than two-molar excess ofthiophenolate, of the two acyl groups, i.e. phenylacetyl and isoxazolylgroups which have been bonded to the nitrogen atom in the 6-position ofpenicillanic acid, only the former is selectively split, and theprotective group of a carboxyl group is simultaneously removedtherefrom.

Thiophenolate used in the above-described reaction is a compound havingthe formula wherein X is hydrogen or a halogen atom and Y is a metalatom, the compound being sodium thiophenolate, sodiumpchlorothiophenorate, potassium thiophenorate, potassiumpchlorothiophenorate or the like.

The isoxazolyl penicillins I obtained according to the present process,e. g. 3-phenyl-5-methyl-4-isoxazolyl penicillin and3-(2'-chlorophenyl)-5-methyl-4-isoxazolyl penicillin, are knownpenicillins. However, they are not only excellent in antibacterialactivity and stable in acids but also resist destruction due topenicillinase and hence are effective against resistant bacteria.Accordingly, they have been used for clinical purposes and are extremelyimportant compounds as antibacterial medicines.

As is clear from the above, the present process, in which the isoxazolylpenicillins l are prepared according to chemical reactions only, withoutusing 6-aminopenicillanic acid but using penicillin G obtained byfermentation, is entirely novel and extremely useful as a process forpreparing the isoxazolyl penicillins I.

The present process will be illustrated in detail below with referenceto particular examples, but the various reaction operations andcompounds which may be employed in the present process are not limitedto those shown in the examples.

EXAMPLE 1 Preparation of benzylpenicillin-p-nitrobenzyl ester:

A mixture comprising 74.5 g. (0.2 mole) of potassium penicillin Gobtained by fermentation and 39.0 g. (0.18 mole) of p-nitrobenzylbromide was added to 500 ml. of dimethylformamide, and the reactionliquid was stirred while maintaining the temperature thereof at 65 C.The progress of the reaction was traced by the iodine method on a silicagel, thin layerchromatogram, and the disappearance of p-nitrobenzylbromide was regarded as the end point of the reaction. After 4 hours,the reaction was substantially complete.

After cooling, the reaction liquid was charged with 300 ml. of benzeneand was washed several times with water, a 10 percent aqueous sodiumcarbonate solution, and water, in this order, to remove the reactionsolvent and unreacted potassium penicillin G, whereby a benzene solutionwas obtained. This solution was dried with anhydrous sodium sulfate andwas then freeze-dried to obtain 63.6 g. of benzylpenicillin-pnitrobenzylester, yield 67.7 percent.

The silica gel, thin layer chromatogram of the above-mentionedfreeze-dried product was obtained with a developing solvent:benzene-ethyl acetate 10:1

Elementary analysis for c l-l O N sz C H N Found 58.93 4.87 8.90Calculated 58.84 4.95 8.94

EXAMPLE 2 Preparation of benzylpenicillin-p-bromophenacyl ester:

Example 1 was repeated, except that p-bromophenacyl bromide was used inplace of the p-nitrobenzyl bromide, to obtainbenzylpenicillin-p-bromophenacyl ester.

Elementary analysis for C ,H O,N SBr:

Found 54.62 4.69 5.49 16.01

Calculated 54.25 4.36 5.27 15.04

EXAMPLE 3 the liquid was reacted for l .5 hours while maintaining thetemperature thereof at 0 C.

After the reaction, deposited pyridine hydrochloride was removed byfiltration, and the reaction mother liquor was quickly washed four timeswith a 1N aqueous sodium bicarbonate solution and twice with water toremove unreacted PCI and byproduct POCI The above-mentioned benzenesolution was dried with ice-cooling with anhydrous sodium sulfate, wasthen immediately charged with 16.9 g. (0.075 mole) of sodium3-phenyl-5-methyl-isoxazol-4-carboxylate, and was stirred at roomtemperature. The progress of the reaction was determined according tothe hydroxamic acid method on a silica gel, thin layer chromatogram, andthe reaction was substantially complete after 20 hours.

The reaction liquid was washed with 0.5N hydrochloric acid, a 1-Naqueous sodium bicarbonate solution, an aqueous saturated sodiumchloride solution, and water, in this order, to remove unreacted3-phenyl-5-methyl-isoxazol-4-carboxylic acid, and the reaction motherliquor was dried with anhydrous sodium sulfate and was then concentratedunder reduced pressure. Subsequently, the concentrate was adsorbed on acolumn comprising silica gel (60-80 mesh; produced by Kanto Kagaku K.K., Tokyo) and was eluted with anhydrous benzeneethyl acetate (10:1),whereby the first eluted fraction contained a desired N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)- benzylpenicillin-pnitrobenzylester. This fraction was recovered and was freeze-dried to obtain 7.51g. of a freezedried product, yield 57.3 percent, [11],, 194 (u'= 1, CHClElementary analysis for C H O,,N S:

Found 62.49 4.57 8.76

Calculated 62.38 4.62 8.56

EXAMPLE 4 Preparation of N-[3-(2-chlorophenyl)-5-methyl-isoxazole-4-carbonyl]-benzy1penicillin-p-nitrobenzyl ester:

Example 3 was repeated, except that sodium 3-(2-chlorophenyl)-5-methyl-isoxazol-4-carboxylate was used in place of thesodium 3-phenyl-5-methyl-isoxazol-4-carboxylate, to obtainN-[3-(2-chlorophenyl)-5-methyl-isoxazole-4-carbonyl]-benzylpenicillin-p-nitrobenzyl ester, yield 51.6 percent.

Elementary analysis for C H O N SCI:

Found 59.43 4.32 8.00

Calculated 59.26 4.24 8.13

EXAMPLE 5 Preparation ofN-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-bromophenacylester:

Example 3 was repeated, except that the benzylpenicillin-pbromo-phenacylester obtained in Example 2 was used in place of thebenzylpenicillin-p-nitrobenzyl ester, to obtain N-(3-phenyl-5-methyl-isoxazole-4-carbony1)-benzylpenicillin-pbromophenacylester, yield 52.3 percent.

Elementary analysis for CQ5H3QO7N3SBII N Br Found 58.47 4.29 5.95 11.00Calculated 58.66 4.22 5.86 11.15

EXAMPLE 6 Elementary analysis for C ,,H O N SClBr:

Preparation of N-( 3-phenyl-5methyl-isoxazole-4-carbonyl)-benzylpenicillin-methyl ester:

Example 3 was repeated, except that benzylpenicillinmethyl ester wasused in place of the benzylpenicillin-p-nitro-benzyl ester, to obtainN-( 3-phenyl-S-methyl-isoxazole-4carbonyl)- benzylpenicillin-methylester, yield 67.1 percent.

Elementary analysis for C H O N S:

H N Found 62.88 5.11 7.79 Calculated 63.02 5.10 7.87

The benzylpenicillin-methyl ester employed in the above reaction hadbeen prepared according to a known process in which potassium penicillinG obtained by fermentation was treated with diazomethane.

EXAMPLE 8 Elementary analysis for C ,,l-l O N SCl:

N Cl Found 59.33 4.63 7.45 6.23 Calculated 59.20 4.61 7.40 6.24

The benxylpenicillin-me'thyl ester employed in the above reaction hadbeen prepared according to a known process in which potassium penicillinG obtained by fermentation was treated with diazomethane.

EXAMPLE 9 Preparation of 3-phenyl-5-methyl-4-isoxazolylpenicillin-pnitrobenzyl ester:

12.5 g. (0.02 mole) of the N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-nitrobenzyl ester was dissolved in 100ml. of dry benzene. To this solution was added dropwise 4.84 ml. (0.04mole) of cyclohexylamine, with stirring, at room temperature. Theprogress of the reaction was determined according to the hydroxamicmethod on a silica gel, thin layer chromatogram, and the reaction wassubstantially complete after 3 hours. According to the above reaction,the phenylacetyl group was cleaved and N-cyclohexyl-phenylacetamide waspresent as a by-product.

The reaction liquid was washed with 0.5N hydrochloric acid and water, inthis order, to remove unreacted cyclohexylamine as a water-solublehydrochloride. The reaction mother liquor was dried with anhydroussodium sulfate and was then concentrated under reduced pressure toobtain an oily substance (about 22.8 g.). To this oily substance wasfurther added dry benzene, and an insoluble precipitate was removed byfiltration using Celite. The filtrate was again concentrated, and theconcentrate was adsorbed on a column comprising 200 g. of silica gel(60-80 mesh) and was subjected to column chromatography usingbenzene-ethyl acetate (5:1) as a developing solvent. Eluted fractionswere traced according to the hydroxamic acid method on a silica gel,thin layer chromatogram, and only the fractions containing 3-phenyl-S-methyl-4-isoxazolyl-penicillin-p-nitrobenzyl ester wererecovered and were freeze-dried to obtain 7.56 g. of a freeze-driedproduct, yield 70.4 percent.

Elementary analysis for C H O,N S:

C H N Found 57.98 4.58 10.52

Calculated 58.19 4.51 10.44

Furthermore, 726 mg. of N-( 3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-nitrobenzyl ester was recovered.

EXAMPLE 10 Preparation of3-(2-chlorophenyl)-5-methy1-4-isoxazolylpenicillin-p-nitrobenzyl ester:

13.1 g. (0.02 mole) of the N-[3-(2'-chlorophenyl)-5-methyl-isoxazole-4-carbonyl]-benzylpenicillin-p-nitrobenzyl ester wasdissolved in ml. of dry benzene. To this solution was added dropwise4.84 ml. (0.04 mole) of cyclohexylamine, with stirring at roomtemperature. The progress of the reaction was determined according tothe hydroxamic acid method on a silica gel, thin layer chromatogram, andthe reaction was substantially complete after three hours. By the abovereaction, the phenylacetyl group was cleaved and N-cyclohexyl-phenylacetamide was present as by-product.

The reaction liquid was washed with 0.5-N hydrochloric acid and water,in that order, to remove unreacted cyclohexylamine as a water-solublehydrochloride. The reaction mother liquor was dried with anhydroussodium sulfate and was then concentrated under reduced pressure toobtain an oily substance. This oily substance was adsorbed on a columncomprising 200 g. of silica gel (60-80 mesh) and was subjected to columnchromatography using benzene-ethyl acetate (5:1) as a developingsolvent. Eluted fractions were traced according to the hydroxamic acidmethod on a silica gel, thin layer chromatogram, and only the fractionscontaining [3-( 2-chlorophenyl)-5-methyl-4-isoxazolyl]-penicillin-p-nitrobenzyl ester wererecovered and were freeze-dried to obtain 7.50 g. of a freeze-driedproduct, yield 66.6 percent.

Elementary analysis for C H O,N SCl:

C H N Cl Found 54.45 4.13 9.67 6.45 Calculated 54.69 4.06 9.81 6.21

EXAMPLE 1 1 Preparation of3-phenyl-5-methyl-4-isoxazolyl-penicillin-pnitrobenzyl ester:

Example 9 was repeated, except that benzylamine was used in place of thecyclohexylamine, to obtain 3-phenyl-5-methyl-4-isoxazolyl-penicillin-p-nitrobenzyl ester, yield 65.8 percent.

Elementary analysis for C H,.,O N S:

N Found 57.99 4.53 10.51 Calculated 58.19 4.51 10.44

EXAMPLE 12 Preparation of3-(2'-chlorophenyl)-5-methyl-isoxazolylpenicillin-p-nitrobenzyl ester:

Example 9 was repeated, except that benzylamine was used in place of thecyclohexylamine, to obtain3-(2-chloro-phenyl)-5-methyl-4-isoxazolyl-penicillin-p-nitrobenzylester, yield 68.4 percent.

Elementary analysis for C H O MSCI:

C H N Cl Found 54.32 4.07 9.72 6.30 Calculated 54.69 4.06 9.81 6.21

EXAMPLE 13 Elementary analysis for C H C N SBr:

C H N Br Found 54.92 4.28 6.92 13.44 Calculated 54.19 4.04 7.02 13.35

EXAMPLE 14 bromophenacyl ester.

Elementary analysis for C H O N SCIBr:

C H N Found 51.92 3.64 6.87 Calculated 51.24 3.66 6.64

EXAMPLE 15 Preparation of3-phenyl-5-methyl-4-isoxazolyl-penicillinmethyl ester;

Example 9 was repeated, except that the N-( 3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-methyl ester obtained inExample 7 was used in place ofthe N-(3-phenyl-5-methyl-isoXazole-4-carbonyl)-benzylpenicillin-p-nitrobenzyl ester, toobtain 3-phenyl-5-methyl-4-isoxazolyl-penicillinmethyl ester, yield 74.5percent.

Elementary analysis for CMHMOSNJS:

C H N Found 58.00 5.21 10.13 Calculated 57.82 5.09 10.11

EXAMPLE 16 Preparation of3-(2'-chlorophenyl)-5-methyl-4-isoxazolylpenicillin-methyl ester:

Example 10 was repeated, except that the N-[3-(2-chlorophenyl)--methyl-isoxazole-4-carbonylJ-benzylpenicillin-methylester obtained in Example 8 was used in place of the N-[ 3-( 2-chlorophenyl )-5-methyl-isoxazole-4-carbonyl]-benzylpenicillin-p-nitrophenyl ester, to obtain 3-(2-chlorophenyl)-5-methyl-4-isoxazolyl-penicillin-methyl ester, yield 68.7percent.

Elementary analysis for C H O N SCI:

Found 53.31 4.55 9.42 8.02 Calculated 53.39 4.48 9.34 7.88

EXAMPLE 1 7 Preparation of 3-phenyl-S-methyl-iosxazolyl-penicillinN,N'-dibenzylethylenediamine salt:

2.68 g. (5 mmol.) of the3-phenyl-5-methyl-4-isoxazolylpenicillin-p-nitrobenzyl ester obtained inExample 9 was dissolved in 30 ml. of dry ethyl acetate. To this solution1.40 ml. 10 mmol.) of triethylamine was added and then the mixture wassubjected to hydrogenation at normal temperature and pressure in thepresence of 3 g. of a 5 percent palladium-active carbon catalyst. Theprogress of the reaction was determined according to the hydroxamic acidmethod on a silica gel, thin layer chromatogram, and the reaction wassubstantially complete after one hour.

The reaction mixture was freed from the catalyst by filtration, and thereaction mother liquor was extracted several times with 30 ml. of water.To this water extract (pH 6.8) was added an aqueous solution ofN,N-dibenzyl-ethylenediamine diacetate to form a water-insoluble salt.The precipitated salt was recovered by filtration, was washed with waterand was then thoroughly dried to obtain 2.56 g. of a powder of3-phenyl-5-methyl-4-isoxazolyl penicillin N,N'-dibenzylethylenediaminesalt, yield 49.0 percent.

The titer of the thus obtained product was substantially identical withthat of a salt of N,N-dibenzyl-ethylenediamine formed from acommercially available oxacillin. According to biological assay, theproduct had a purity of 94 percent.

Elementary analysis for (C,,,H, O,,N,S),-C H, N,:

C H N Found 62.53 5.67 10.70 Calculated 62.17 5.60 10.74

Thus, the 3-phenyl-5-methyl-4-isoxazolyl penicillin prepared accordingto the present process coincides both physically and biologically withthat prepared according to the conventional process.

EXAMPLE 18 Preparation of 3-phenyl-5-methyl-4-isoxazolyl-penicillinsodium:

2.82 g. (5 mmol.) of the3-pheny1-5-methyl-4-isoxazolylpenicillin-p-bromophenyl ester wasdissolved in 25 ml. of dimethylformamide. To this solution was added1.32 g. (10 mmol.) of sodium thiophenoxide, and the mixture was reactedat room temperature for 20 minutes.

Subsequently, the reaction mixture was charged with 500 ml. of acetoneand was stirred, and the precipitate was recovered by filtration, waswashed with acetone and was then dried to obtain 1.42 g. of sodium3-phenyl-5-methyl-4-isoxazolyl-penicillin, yield 67.1 percent.

The titer of the thus-obtained product coincided with that of acommercially available sodium oxacillin. According to biological assay,this product had a purity of 95 percent.

Elementary analysis for C,,,H, O .,N,SNa:

Found 54.33 4.34 9.75

Calculated 54.89 4.28 9.92

EXAMPLE 19 Preparation of3-(2-chlorophenyl)-5-methyl-4-isoxazolylpenicillin N,N-dibenzyl-ethylenediamine salt:

2.86 g. (5 mmol.) of the 3-(2-chlorophenyl)-5-methyl-4-isoxazolyl-penicillin-p-nitrobenzyl ester was dissolved in 30 ml. of dryethyl acetate. To this solution was added 1.40 ml. 10 mmol.) oftriethylamine, and the mixture was subjected to hydrogenation at nonnaltemperature and pressure in the presence of 3 g. of a 5 percentpalladium-active carbon catalyst. The progress of the reaction wasdetermined according to the hydroxamic acid method on a silica gel, thinlayer chromatogram, and the reaction was substantially complete after 1hour.

The reaction mixture was freed from the catalyst by filtration, and thereaction mother liquor was extracted several times with 30 ml. of water.To this extract (pH 6.8) was added an aqueous solution of N,N'-dibenzylethylenediamine diacetate to form a water-insoluble salt. Theprecipitated salt was recovered by filtration, was washed with water andwas then thoroughly dried to obtain 2.48 g. of3-(2'-chlorophenyl)-5-methyl-4-isoxazolyl-penicillinN,N-dibenzylethylenediamine salt, yield 44.6 percent. According tobiological assay, the product had a purity of 92.5 percent.

Elementary analysis for (C l-l O N SCl),C, H, N

C H N penicillin prepared according to the present process coincidesboth physically and biologically with that prepared according to theconventional process.

EXAMPLE 20 Preparation of sodium 3-(2-chlorophenyl)-5-methy1-4-isoxazolyl-penicillin:

Elementary analysis for C l-l ,O,N,SClNa:

C l-l N Cl Found 53.08 4.20 9.79 8.23 Calculated 53.59 4.02 9.87 8.33

The thus-obtained product coincided both physically and biologicallywith that prepared according to the conventional process.

EXAMPLE 21 Preparation of 3-pheny1-5-methyl-4-isoxazo1yl-penicillinsodium salt:

35.84 g. (0.05 mole) of the N-( 3-phenyl-5-methyl-4-isoxazolyl)-benzyl-penicillin-p-bromophenacyl esterwas dissolved in 100 ml. of dry chloroform. To this solution was added7.39 ml. (0.075 mole) of n-butylamine at room temperature with stirring.The reaction progress was followed according to the hydroxamic acidmethod on a silica gel, thin layer chromatogram, and the reaction wassubstantially complete after three hours.

The phenylacetyl group was split to produce N-N-butylphonylacetamide asa byproduct in accordance with the above-described reaction.

The reaction mixture hereinabove was washed with 0.5 N-HCl and waterrespectively to remove unreacted n-butylamine as a hydrochloride. Afterdrying the reaction mixture with anhydrous sodium sulfate, the saidmother liquor was concentrated in vacuo. The yielded oily substance wasdissolved in dry benzene, and charged on a column of silica gel (500 g.,60-80 mesh), then eluted with benzene-ethylacetate (5:1 1). The eluate,containing 3-phenyl-5-methyl-4-isoxazoly1 penicillin-p-bromophenacylester, which was detected by the hydroxamic acid method on a silica gelthin layer chromatogram, was collected and concentrated in vacuo toobtain a condensate.

The said condensate was dissolved in 75 ml. of dimethylformamide, slowlyadding 6.6 g. (0.05 mole) of sodium thiophen- Elementary analysis for CH O N SNa:

C l-l N Found 54.89 4.28 9.92 Calculated 55.14 4.12 9.77 Purity: 81% bybioassay EXAMPLE 22 Preparation of sodium 3-phenyl-5-methyl-4-isoxazolylpenicillin:

35 .84 g. (0.05 mole) of N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-bromophenacyl ester was dissolved in 50 ml.of dry chloroform. To the solution 12 g. (0.065 mole) of laurylamine in50 ml. of dry chloroform was dropwise added with stirring for 7 hours.The reaction was continued for an additional hour. 100 ml. of drybenzene and 15 g. of silica gel were added thereto. After stirring for40 hours, the solution was filtered and the filtrate was concentrated invacuo to remove chloroform. The concentrate was charged on a g. ofsilica gel column in benzene (-80 mesh) and eluted withbenzene-ethylacetate (10:1) to elute 3- phenyl-5-methyl-4isoxazolylpenicillin-p-bromophenacyl ester, which was concentrated in vacuo toobtain a condensate. Sodium thiophenoxide was reacted with the saidcondensate to remove the ester by the same procedure as in Example 21.3-phenyl-5-methyl4-isoxazolyl penicillin sodium salt was yielded as alyophilizate.

Yield: 9.8 g. (recovery: 50.1 percent) Purity: 92 percent by bioassayEXAMPLE 23 Preparation of 3-phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

35.84 g. (0.05 mole) of N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-bromophenacyl ester was dissolved in 100ml. of dry chloroform. To this solution 19.25 g. (0.075 mole) ofstearylamine in 80 ml. of dry chloroform was dropwise added withstirring at 0 C.

After 3 hours of reaction, the reaction mixture was charged on a columnof 1 kg. of silica gel (60-80 mesh) in chloroform and was eluted withbenzene-chloroform 1: 1

The elute was analyzed according to the hydroxamic acid method on asilica gel, thin layer chromatogram, and a fraction containing3-phenyl-5-methyl-4-isoxazolyl penicillin-pbromophenacyl ester thereinwas collected, and then concentrated in vacuo to obtain a concentrate.

De-esterification was achieved according to Example 21 by sodiumthiophenoxide to yield 3-pheny1-5-methyl-4-isoxazolyl penicillin sodiumsalt as a lyophilizate.

Yield: 10.0 g. (recovery 51.1 percent) Purity: 95 percent by bioassayEXAMPLE 24 Preparation of 3-(2-chlorophenyl)-5-methyl-4-isoxazolylpenicillin sodium salt:

In Example 21,N-(3-phenyl-S-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-bromophenacylester was replaced by N[3-(2-chlorophenyl)-5methyl-isoxazole-4-carbonyl]-benzylpenicillin-p-bromophenacyl ester obtained in Example 6.3(2'-chlorophenyl)-5-methyl-4-isoxazolyl penicillin sodium salt wasyielded.

Yield: 47.8 percent Purity: 78.5 percent by bioassay EXAMPLE 25Preparation of 3-(2-chlorophenyl)-5-methyl-4-isoxazolyl penicillinsodium salt.

In Example 22,N-(3-pheny1-5-methyl-isoxazole-4-carbonyl)-benzy1penicillin-p-bromophenacylester was replaced by N-[ 3-(2'-chlorophenyl)-5-methyl-isoxazole-4-carbonyl1-benzylpenicillin-p-bromophenacyl ester to produce 3-(2'-chlorophenyl)-5-methyl-4-isoxazolyl penicillin sodium salt.

Yield: 45.4 percent Purity: 91.5 percent by bioassay EXAMPLE 26Preparation of 3-(2-ch1orophenyl)-5-methyl-4-isoxazolyl penicillinsodium salt: charged 37.55 g. (0.05 mole) ofN-[3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl]-benzylpenicillin-p-bromophenacylester obtained in Example 6 was dissolved in 40 ml. of dry chloroform.To this solution 18.9 g. (0.07 mole) of stearylamine dissolved in ml. ofdry chloroform was dropwise added with stirring for 30 minutes and thereaction was continued for about 1.5 hours. The reaction mixture waschared on a column of silica gel (600 g., 60-80 mesh) in chloroform andwas eluted with a mixed solution of benzenechloroform (ratio 1:1). Thefraction containing 3-(2'- chlorophenyl)-5-methyl-4-isoxazolylpenicillin-pbromophenacyl ester, which was eluted afier elution ofbyproduct N-stearyl-phenylacetamide, was collected and was concentratedin vacuo to obtain a concentrate.

The de-esterfication was carried out according to the process of Example21 to obtain 3-(2-chlorophenyl)-5- methyl-4-isoxazolyl penicillin sodiumsalt as a lyophilizate.

Yield: 9.2 g. (recovery: 43.2 percent) Purity: 92.8 percent by bioassayEXAMPLE 27 Preparation of 3-phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

32.7 g. (0.05 mole) of N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-nitrobenzyl ester obtained in Example 3 wasdissolved in 100 ml. of dry chloroform. To this solution 19.25 g. (0.075mole) of stearylamine dissolved in 80 ml. of dry chloroform was dropwiseadded with stirring at C. After 3 hours reaction, the reaction mixturewas charged on a column of silica gel (1 kg., 60-80 mesh) in chloroformand was eluted therefrom with a mixed solution of benzenechloroform1:1).

The fraction containing 3-phenyl-5-methyl-4-isoxazolylpenicillin-p-nitrobenzyl ester was collected and concentrated in vacuo.The concentrate was dissolved in 180 ml. of dry ethylacetate and thesolution was hydrogenized in the presence of 20 g. of percentpalladium-charcoal at room temperature. The progress of the reaction wasfollowed according to the hydroxamic acid method on a silica gel thinlayer chromatogram. Accordingly the reaction was completed within 1hour.

After the reaction mixture hereinabove was filtered to remove catalyst,a mother liquor was extracted with 5 percent solution of sodiumbicarbonate and phosphoric acid was added thereto until pl-l 2 wasreached, followed by extraction with methyl isobutyl ketone. Afterwashing the methyl isobutyl ketone layer with water, reextraction with asmall amount of sodium bicarbonate solution was repeated and thethus-obtained extract was lyophilized to yield 3-phenyl-5-methyl-4-isoxazolyl penicillin sodium salt.

Yield: 6.9 g. (recovery: 35.3 percent) Purity: 92.5 percent by bioassayElementary analysis for C H- O N SNa:

Preparation of 3-(2'-chlorophenyl)-5-methyl-4-isoxazolyl penicillinsodium salt:

35.45 g. (0.05 mole) ofN-[3-(2'-chlorophenyl)-5-methylisoxazole-4-carbonyl]-benzylpenicillinsodium salt was dissolved in 50 ml. of dry chloroform. 18.9 g. (0.07mole) of stearylamine dissolved in 150 ml. of dry chloroform wasdropwise added at 0 C. with stirring for about 30 minutes. After 1 hoursreaction, the reaction mixture was charged on a column of silica gel(600 g., 60-80 mesh) in chloroform and eluted with a mixed solution ofbenzene-chloroform (1:1). The fraction containing3-(2'-chlorophenyl)-5-methyl-4-isoxazolyl penicillin-p-nitrobenzyl esterwas collected, followed by concentration in vacuo. The hydrogenation ofthe said concentrate was achieved in accordance with the processdescribed in Example 27 to obtain3-(2-chlorophenyl)-5-methyl-4-isoxazolyl penicillin sodium slat as alyophilizate.

Yield: 31 percent EXAMPLE 29 Preparation of3-phenyl-5-methyl-4-isoxazolyl penicillinphenacyl ester:

31.9 g. (0.05 mole) of N-(3-phenyl-S-methyl-isoxazole-4- carbonyl)-benzylpenicillin phenacyl ester was dissolved in 165 ml. of distilledacetone. To this solution 6.93 g. (0.0525 mole) of sodium thiophenolatein 15 ml. of dimethylformamide was dropwise added. After the reactioncontinued for 30 minutes, 130 ml. of benzene was added thereto andwashed with 250 ml. of 1 N HCl (pH 2) and 250 ml. of water in that orderto remove unreacted thiophenolate and dimethylformamide. The reactionmixture was washed with l N sodium bicarbonate solution for three timesto remove by-product 3-phenyl-5-methyl-4-isoxazolyl penicillin. Themixture was also washed twice with 60 ml. of water. After dehydration byanhydrous sodium sulfate, the reaction mixture was concentrated and thenwashed with petroleum ether three times to remove phenylacetatethiophenyl ester.

The thus-obtained crystalline compound was filtered and dried to yield3-phenyl-5-methyl-4-isoxazolyl penicillinphenacyl ester.

Yield: 17.8 g. (recovery: 67.1 percent) Elementary analysis for C H O NS:

Found 62.47 4.83 8.06

Calculated 62.41 4.87 8.09

EXAMPLE 30 Found 58.21 4.40 7.49

Calculated 58.53 4.36 7.54

EXAMPLE 31 Preparation of 3-(2,6-dichloropehnyl)-5-methyl-4-isoxazolylpenicillin phenacyl ester:

In Example 29, 3-phenyl-5-methyl-isoxa2ole-4-carbonylbenzyl-penicillinphenacyl ester was replaced by 3-(2',6'-dichlorophenyl)-5-methyl-isoxazole-4-carbonyl benzylpenicillin phenacylester to produce 3-(2,6-dichlorophenyl)-5- methyl-4-isoxazolylpenicillin phenacyl ester.

Yield: 49.5 percent Elementary analysis for C l-l O N sclz C H N Found54.90 3.91 7.09 Calculated 55.10 3.93 7.14

EXAMPLE 32 Preparation of 3phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

26.0 g. (0.05 mole) of 3-pheny1-5-methyl-4-isoxazolyl penicillinphenacyl ester obtained in Example 29 was dissolved in ml. of distilleddimethylformamide. To this solution 7.62 g. (0.06 mole) of sodiumthiophenolate in 15 ml. of dimethylformamide was dropwise added withstirring at 0-3 C. over 15 minutes. After 30 minutes reaction,butylacetate ml.) was added thereto, followed by washing with 250 ml. of1 N HCl (pH 2) and 250 ml. of water, in that order, to remove unreactedthiophenolate and dimethylformamide, respectively. The butylacetatelayer was extracted twice with 30 ml. of l N sodium bicarbonate solutionand water. The water layer and sodium bicarbonate solution layer werecombined and extracted twice with 40 ml. of ethylacetate after adjustingthe solution to pH 2 with l N l-lCl. After the ethylacetate layer waswashed with water and dehydrated by anhydrous sodium sulfate, 10 g. ofsodium 2-ethyl hexanoate in dry ethylacetate (35 m1.) was added thereto.The solution was concentrated in vacuo, filtered and washed withethylacetate to obtain 3-phenyl-5-methyl-4-isoxazo1yl penicillin sodiumsalt.

Yield: 12.1 g. (recovery: 55.2 percent) M.p.: l93-196 C. (decomposed)Potency (bioassay): 860 mcg./mg. (purity: l percent) EXAMPLE 33Preparation of 3-(2-chlorophenyl)-5-methyl-4-isoxazolyl penicillinsodium slat:

In Example 32, 3-phenyl-5-methyl-4-isoxazolyl penicillin phenacyl esterwas replaced by 3-(2'-chlorophenyl)-5-methyl- .4-isoxazolyl penicillinphenacyl ester obtained in Example 30 to produce3-(2-ch1orophenyl)-5-methyl-4-isoxazolyl penicillin sodium salt.

Yield: 48.8 percent M.p.: l94"l97 C. (decomposed) Potency (bioassay):930 mcgJmg.

Elementary analysis fro C H O MSNaCI'H O C H N Found 47.75 4.03 8.87Calculated 47.95 4.02 8.83

EXAMPLE 34 Elementary analysis for C,,,H, O,N SNaCl -H O Found 45.033.51 8.11

Calculated 44.72 3.55 8.23

EXAMPLE 35 Preparation of 3-phenyl-5-methyl-4-isoxazolylpenicillin-pnitrobenzyl ester:

p 34.5 g. (0.05 mole)v of N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-nitrobenzyl ester was dissolved in 170 ml.of distilled acetone. To this solution 9.9 g. (0.075 mole) of sodiumthiophenolate in distilled dimethylformamide (25 ml.) was added dropwiseat 0-3 C. with stirring, over 15 minutes. After 30 minutes reaction, 140ml. of benzene was added thereto. The mixture was washed with l N HCl(250 ml., pH 2) and water (250 ml.) to remove unreacted thiophenolateand dimethylformamide. The organic solvent layer was washed with 1 Nsolution of sodium bicarbonate and water, in that order, then dehydratedwith anhydrous sodium sulfate, concentrated in vacuo and washed withpetroleum ether 3 times to remove phenylacetate thiophenyl ester. Theconcentrate was dissolved in dry benzene and lyophilized to obtain theproduct.

Yield: 15.1 g. (recovery: 57.0 percent) M.p.: 64-70 C.

Elementary analysis for C H O N S:

C H N Found 58.09 4.48 10.51 Calculated 58.20 4.50 10.44

EXAMPLE 36 Preparation of 3-(2'-chlorophenyl)-5-methyl-4-isoxazolylpenicillin-p-nitrobenzyl ester:

In Example 35,N-(3-phenyl-5-methyl-isoxazole-4-carbonyl)-benzylpenicillin-p-nitrobenzylester was replaced by N-]3- (2'-chlorophenyl)-5-methyl-isoxazole-4-carbonyl]-benzylpenicillin-p-nitrobenzyl ester toproduce the product hereinabove.

Yield: 50.3 percent Elementary analysis for C I-I O-N SCI:

Found 54.33 4.02 9.78 6.27

Calculated 54.68 4.04 9.81 6.20

EXAMPLE 37 Yield: 47.8 percent Elementary analysis for C,.,H,,O,N,SC1,:

C H N Cl Found 52.10 7.49 9.27 10.89 Calculated 51.51 7.66 9.25 l 1.71

EXAMPLE 3 8 Preparation of 3-phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

15.6 g. (0.028 mole) of3-phenyl-5-methyl-4-isoxazolylpenicillin-p-nitrobenzyl ester wasdissolved in ml. of dry ethylacetate. The solution was hydrogenated inthe presence of 5 percent palladium-charcoal catalyst at roomtemperature under atmospheric pressure until no more hydrogen wasabsorbed (about 3 hours).

After washing twice with water, the organic solvent layer was extractedtwice with a l N solution of sodium bicarbonate (30 ml.) and againwashed twice with water (60 ml.). The extract and washing solution werecombined, adjusted to pH 2 with 1 N HCl and extracted twice again with40 ml. of ethylacetate. After the ethylacetate layer was washed withwater and dehydrated with anhydrous sodium sulfate, 8.3 g. of sodium2-ethyl hexanoate in dry ethyl acetate (30 ml.) was added thereto. Thesolution was concentrated in vacuo, filtered to remove precipitatetherefrom then washed with dry ethylacetate to obtain the product.

Yield: 6.70 g. (recovery: 52.3 percent) Potency (bioassay): 840 meg/mg.(purity: 97.7 percent) Elementary analysis for C H O N SNa'H O Found51.45 4.63 9.50

Calculated 51.70 4.57 9.52

EXAMPLE 39 Preparation of 3-(2-chlorophenyl)-5-methyl-4-isoxazolylpenicillin sodium salt:

In Example 38, 3-phenyl-5-methyl-4-isoxazo1y1penicillin-pnitrobenzylester was replaced by 3-(2-chlorophenyl)-5- methyl-4-isoxazolylpenicillin-p-nitrobenzyl ester obtained in Example 36 to produce theproduct hereinabove.

Yield: 48.2 percent Potency (bioassay): 915 meg/mg.

Elementary analysis for C H,,O N SNaCl-H O C H N Found 48.06 3.64 8.71Calculated 47.96 3.60 8.83

EXAMPLE 40 Elementary analysis for C I-l O N SNaCl 'H O N Found 45.063.51 8.18 Calculated 44.72 3.55 8.23

EXAMPLE 41 Preparation ofN-(3-phenyl-5methyl-isoxazole-4-carbonyl)-benzylpenicillin phenacylester: 0.105 continued 30 45.2 g. (0.1 mole) of benzylpenicillinphenacyl ester was dissolved in dry benzene (200 ml.) After adding 32.4ml. (0.4 mole) of dry pyridine at 7 C. with stirring, 21.8 g. (0.015mole) of PCl,, in dry benzene (250 ml.) was dropwise added for about 30minutes. The reaction was continued for about 1.5 hours at 5 to 7 C. Theprecipitated pyridine hydrochloride was removed by filtration, and thereaction mixture was washed with a l N solution of sodium bicarbonateand water to remove unreacted PCl, and by-product POCl After dehydrationwith anhydrous sodium sulfate, 3-phenyl-5-methyl-isoxazolyl-4-carboxylic acid (24.1 g., 0.1 mole) was added to thebenzene layer. The reaction was continues at 30 -35 C. for 4 hours andat 45 C. for 1 hour. The reaction mixture was washed with 0.5 N HCl, 1 Nsodium bicarbonate solution, saturated sodium chloride and water, inthat order, to remove unreacted pyridine and3-phenyl-S-methyl-isoxazolyl- 4-carboxylic acid. After dehydration withanhydrous sodium sulfate, the mother liquor was concentrated in vacuo.The said concentrate was charged on a column of silica gel (150 g.,60-80 mesh) and eluted with anhydrous benzene-ethylacetate solution(20:1).

The first eluate contained the desired product.

Yield: 41.3 g. (recovery: 64.8 percent) as a lyophilizate Elementaryanalysis for C ,,H O,N S:

Found 65.78 4.75 6.78

Calculated 65.93 4.90 6.59

EXAMPLE 42 Elementary analysis for C H O,N -,SCl:

C H N Found 62.95 4.78 6.17 Calculated 62.55 4.50 6.25

EXAMPLE 43 Preparation of 3-phenyl-5methyl-4-isoxazolyl penicillinsodium salt:

31.9 g. (0.05 mole) of N-(3-phenyl-S-methyl-isoxazole-4-carbonyl)-benzylpenicillin phenacyl ester was dissolved in 100 ml. ofdistilled dimethylformamide. To this solution 14.5 g. (0.1 1 mole) ofsodium thiophenolate in distilled dimethylformamide (30 ml.) wasdropwise added with stirring at C. After 30 minutes reaction, 120 ml. ofbutylacetate was added therein and washed with 250 ml. of 1 N HCl and250 ml. of water to remove unreacted thiophenolate anddimethylformamide, respectively. The solvent layer was extracted twicewith 60 ml. of l N sodium bicarbonate solution and washed twice with 60ml. of water. After the said extracts and washings were combined andadjusted to pH 2, the solution was extracted twice with ethylacetate 120ml.) The ethylacetate layer was washed with water, followed bydehydration with anhydrous sodium sulfate, and 8.3 g. of 2-ethylhexanoate in dry ethylacetate (30 ml.) was added therein. The solutionwas concentrated in vacuo, then precipitate was collected by filtration.

Yield: 10.33 g. (recovery: 46.8 percent) Potency (bioassay): 860 meg/mg.(purity: 100 percent) Elementary analysis for C,,,H,,,O,,N SNa'H O C H NFound 51.74 4.44 9.47 Calculated 51.70 4.57 9.52

EXAMPLE 44 Preparation of 3phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

In Example 43, the conditions of the reaction between N-3-phenyl-5methy1-isoxazole-4-carbonyl )-benzylpenicillin phenacyl esterand sodium thiophenolate were replaced by the conditions 5 to 0 C.temperature and 1 hour duration to yield the product.

Yield: 7.28 g. (recovery: 33.0 percent) Potency (bioassay): 860 meg/mg.(purity: percent) EXAMPLE 45 Elementary analysis for C ,H ,O,,N SNa-l-lO Found 51.46 4.62 9.51

Calculated 51.70 4.57 9.52

EXAMPLE 46 Preparation of 3phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

In Example 41, dimethylfonnamide was replaced by acetone to produce thedesired product.

Yield: 31.1 percent Potency (bioassay): 850 meg/mg. (purity: 98.8percent) EXAMPLE 47 Preparation of3-(2-chlorophenyl)-5-methyl-4-isoxa2olyl penicillin sodium salt:

33.6 g. (0.05 mole) ofN-[3-(2chlorophenyl)-5-methylisoxazole-4-carbonyl] benzylpenicillinphenacyl ester was dissolved in 100 ml. of distilled ethylacetate. Tothis solution, 14.5 g. (0.11 mole) of sodium thiophenolate indimethylformamide (30 ml.) was dropwise added with stirring at -5 to 0C. over 15 minutes. After 1 hour, the reaction mixture was washed with130 ml. of 1 N HCl and 130 ml. of water to remove unreactedthiophenolate. The ethylacetate layer was extracted twice with 60 ml. ofl N sodium bicarbonate solution and washed twice with 60 ml. of water.The solution was adjusted to pH 2 with l N HCl, then extracted twicewith ml. of ethylacetate. The thus-extracted layer was washed with waterand dehydrated with anhydrous sodium sulfate; 8.3 g. of sodium2-ethylhexanoate in dry ethylacetate (30 ml.) were added thereto and themixture was concentrated in vacuo. The precipitated material wascollected, washed with ethylacetate and dried in vacuo to yield3-(2-chlorophenyl)- 5-methyl-4-is0xazolyl penicillin sodium salt.

Yield: 8.49 g. (recovery: 37.6 percent) Potency (bioassay): 950 meg/mg.

Elementary analysis for C H O N SNaCl-H O C H N Found 48.18 3.69 8.74Calculated 47.96 3.60 8.83

EXAMPLE 48 Preparation of 3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolylpenicillin sodium salt:

35.3 g. (0.05 mole) of N-[3-(2,6-dichlorophenyl)-5- methyl-isoxazole-4carbonyl]-benzylpenicillin phenacyl ester was dissolved in 100 ml. ofdimethylformamide. To this solution 14.5 g. (0.11 mole) of sodiumthiophenolate in dimethylformamide (30 ml.) was dro'pwise added withstirring at 5 to C. over 15 minutes. After 1 hour, the reaction mixturewas treated by the same procedure as in Example 47 to produce thedesired product.

Yield: 4.67 g. (recovery: 18.3 percent) Potency (bioassay): 910 meg/mg.

Found 45.10 3.53 8.16 Calculated 44.72 3.55 8.23

EXAMPLE 49 Preparation of 3-phenyl-5-methyl-4-isoxazolyl penicillinsodium salt:

35.8 g. (0.05 mole) of N-(3phenyl-5-methyl-isoxazole-4- carbonyl)benzylpenicillin-p-bromophenacyl ester was dissolved in 100 ml. ofdimethylformamide. To this solution 14.5 g. (0.1 1 mole) of sodiumthiophenolate in dimethylformamide (30 ml.) was dropwise added at to 0C. with stirring over 15 minutes. After 1 hour, the reaction mixture wastreated by the same procedure as in Example 43 to obtain the desiredproduct.

Yield: 33.0 percent Potency (bioassay): 855 mcg./mg. (purity: 99.4percent) EXAMPLE 50 Preparation of3-(2-chlorophenyl)-5-methyl-4-isoxazolyl penicillin sodium salt:

37.6 g. (0.05 mole) ofN-[3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl]benzylpenicillimp-bromophenacylester was dissolved in 100 ml. of dimethylformamide. To this solution14.5 g. (0.11 mole) of sodium thiophenolate in dimethylformamide (30ml.) was dropwise added with stirring at -5 to 0 C. over 15 minutes.After 1 hour, the reaction mixture was treated by the same procedure asin Example 43 to obtain 3- (2'-chloro-phenyl)-5-methyl-4-isoxazolylpenicillin sodium salt.

Yield: 30.5 percent Potency (bioassay): 940 meg/mg.

Having described our invention we claim:

l. A process for the production of a member selected from the groupconsisting of isoxazolyl penicillins having the formula and nontoxicsalts and lower alkyl esters thereof, wherein R is a member selectedfrom the class consisting of hydrogen and lower alkyl and R, and R areeach a member selected from the group consisting of hydrogen andhalogen, comprising reacting a penicillin G ester having the formulawherein R is a protective group for carboxyl group and is selected fromthe group consisting of lower alkyl of C pmethoxybenzyl, benzyl,p-nitrobenzyl, benzhydril, phenacyl, p-bromophenacyl and trimethylsilyl,with a chlorinating agent selected from the group consisting of PCI,,,PCl,, PO0

CQC1 and SOC1,, in the presence of pyridine to obtain an imide chloridegroupnncorporated compound having the forwherein R is as defined above;reacting the compound of F ormula III with an isoxazol carboxylatehaving the formula wherein M is a metal atom, and R and R are as definedabove, to obtain a diacyl penicillin ester having the formula chloridegroup-incorporated compound of Formula III is used in the form of amixture of 1 mole thereof with l-2 moles of isoxazol carboxylate ofFormula IV, and the reaction is carried out at a temperature of 20 to 50C.

4. A process according to claim 1, wherein the diacyl penicillin esterof Formula V is used in the form of a mixture of 1 mole thereof with l-2moles of aminolyzing agent selected from the group consisting of primaryorganic amine and ammonia, and the reaction is carried out at atemperature of 5 to 40 C.

5. A process according to claim 4, wherein the aminolyzing agent ispyridine.

6. A process according to claim 1, wherein the diacyl penicillin esterof Formula V is used in the form of a mixture of 1 mole thereof with l-4moles of thiophenolate, and the reaction is carried out at a temperatureof -5 to 40 C.

2. A process according to claim 1, wherein the carboxyl group protectedpenicillin G is in the form of a mixture of 1 mole thereof with 1-2moles of chlorinating agent in the presence of 1-5 moles of pyridine,and the reaction is carried out at a temperature of -20* to 25* C.
 3. Aprocess according to claim 1, wherein the imide chloridegroup-incorporated compound of Formula III is used in the form of amixture of 1 mole thereof with 1-2 moles of isoxazol carboxylate ofFormula IV, and the reaction is carried out at a temperature of 20* to50* C.
 4. A process according to claim 1, wherein the diacyl penicillinester of Formula V is used in the form of a mixture of 1 mole thereofwith 1-2 moles of aminolyzing agent selected from the group consistingof primary organic amine and ammonia, and the reaction is carried out ata temperature of -5* to 40* C.
 5. A process according to claim 4,wherein the aminolyzing agent is pyridine.
 6. A process according toclaim 1, wherein the diacyl penicillin ester of Formula V is used in theform of a mixture of 1 mole thereof with 1-4 moles of thiophenolate, andthe reaction is carried out at a temperature of -5* to 40* C.